The mechanism of heparin stimulation of rat adipocyte lipoprotein lipase

Free fat cells and stromal-vascular cells were prepared from rat adipose tissue by incubation with collagenase. NH(4)OH-NH(4)Cl extracts of acetone-ether powders prepared from fat cells contained lipoprotein lipase activity but extracts of stromal-vascular cells did not. Intact fat cells released lipoprotein lipase activity into incubation medium, but intact stromal-vascular cells did not. The lipoprotein lipase activity of the medium was increased when fat cells were incubated with heparin, and this was accompanied by a corresponding decrease in the activity of subsequently prepared fat cell extracts. Heparin did not release lipoprotein lipase activity from stromal-vascular cells. The lipoprotein lipase activity of NH(4)OH-NH(4)Cl extracts of fat cell acetone powders is increased by the presence of heparin during the assay. This increase is not due to preservation of enzyme activity, but to increased binding of lipoprotein lipase to chylomicrons. Protamine sulfate and sodium chloride have little effect on the binding of lipoprotein lipase to chylomicrons, but they inhibit enzyme activity after binding to substrate has occurred. These inhibitors do, however, inhibit the stimulatory effect of heparin on enzyme-substrate binding.     

Distribution of lipoprotein lipase and hepatic lipase between plasma and tissues: effect of hypertriglyceridemia

Lipoprotein lipase and hepatic lipase were measured in rat plasma using specific antisera. Mean values for lipoprotein lipase in adult rats were 1.8-3.6 mU/ml, depending on sex and nutritional state. Values for hepatic lipase were about three times higher. Lipoprotein lipase activity in plasma of newborn rats was 2-4-times higher than in adults. In contrast, hepatic lipase activity was lower in newborn than in adult rats. Following functional hepatectomy there was a progressive increase in lipoprotein lipase activity in plasma, indicating that transport of the enzyme from peripheral tissues to the liver normally takes place. Lipoprotein lipase, but not hepatic lipase, increased in plasma after a fat meal. An even more marked increase, up to 30 mU/ml, was seen after intravenous injection of Intralipid. Plasma lipase activity decreased in parallel with clearing of the injected triacylglycerol. 125I-labeled lipoprotein lipase injected intravenously during the hyperlipemia disappeared somewhat slower from the circulation than in fasted rats, but the uptake was still primarily in the liver. Hyperlipemia, or injection of heparin, led to increased lipoprotein lipase activity in the liver. This was seen even when the animals had been pretreated with cycloheximide to inhibit synthesis of new enzyme protein. These results suggest that during hypertriglyceridemia lipoprotein lipase binds to circulating lipoproteins/lipid droplets which results in increased plasma levels of the enzyme and increased transport to the liver.     

Hormonal mediation of rat heart lipoprotein lipase activity after fat feeding

The effect of acute fat feeding on the response of two fractions of lipoprotein lipase in heart was explored. In rats, previously fasted, lipoprotein lipase activity released into the perfusate by heparin increased approximately 50% 4 h after fat feeding. The lipase activity remaining in the heart tissue after heparin perfusion showed no significant difference. When rats maintained ad libitum were intubated with glucose 2 h before the fat dose, a relatively larger increase (5-10-fold) in the heparin-releasable lipase activity was observed. The capacity of these hearts to hydrolyze 14C-labeled chylomicrons was also increased 4-5-fold over the controls. Fat ingestion has been reported to elevated plasma corticosteroid levels in rats. When adrenalectomized rats were fed fat, no significant changes in the heparin-releasable lipase activity were observed Hydrocortisone and corticotropin treatment increased the heparin-releasable lipase activity to the same degree as observed with fat feeding. These data suggest that the increase in heart lipoprotein lipase activity following fat feeding is mediated via corticosteroids.     

Hepatic and extrahepatic uptake of intravenously injected lipoprotein lipase

Rats were injected intravenously with 125I-labeled bovine lipoprotein lipase. The lipase disappeared within minutes from the blood due to uptake both in the liver (about 50% of the injected dose) and in extrahepatic tissues. Lipase enzyme activity disappeared in parallel to the 125I radioactivity. Thus, there was no inactivation of lipase in the circulating blood. Similar results were obtained when lipoprotein lipase purified from guinea pigs was injected into guinea pigs. Using supradiphragmatic rats we could show that the extrahepatic uptake was saturable and that the amounts of lipase that could be bound far exceeded the amounts of endogenous lipase expected to be present on the endothelium. When the lipase was denatured before injection, its removal in supradiaphragmatic rats became slower, and in intact rats the fraction of the uptake that occurred in extrahepatic tissues was much decreased. It is concluded that recognition by the extrahepatic receptors depends on the native conformation of the lipase. The extrahepatic uptake was strongly impeded by injection of heparin prior to injection of the lipase, and the uptake could to a large extent be reversed by injection of heparin after the lipase. Even after 1 h lipase that had been taken up by extrahepatic tissues reappeared immediately in the blood on injection of heparin. This was true both for enzyme activity and for enzyme radioactivity. Thus, internalization-inactivation-degradation occur only slowly in extrahepatic tissues. It is possible that the extrahepatic binding occurs to the enzyme's physiological receptors. The hepatic uptake was not dependent on the native conformation of the lipase, was less sensitive to heparin, could not be reversed by heparin and was not saturable. The enzyme was not rapidly inactivated after uptake; its activity could be detected in liver homogenates even after 1 h. Degradation to acid-soluble products in the liver was relatively slow; the t1/2 for native lipase was about 1 h. In comparison, in parallel experiments asialofetuin was degraded with a t1/2 of about 15 min.     

Activity of lipoprotein lipase in thyroidectomized rats

Total plasma postheparin lipolytic activity as well as lipoprotein lipase activity in plasma was higher after heparin injection in thyroidectomized rats than in controls. In contrast, the activity of liver lipase was lower in thyroidectomized rats. Adipose tissue from thyroidectomized rats contained more lipoprotein lipase activity than adipose tissue from controls as measured both in extracts of tissue homogenates and medium from in vitro incubations of tissue pieces. There were no differences between control and hypothyroid rats in the disappearance of intravenously injected 125I-labeled lipoprotein lipase, but when a low dose of heparin was injected before the labeled enzyme, the disappearance of 125I-labeled lipoprotein lipase was more retarded in thyroidectomized rats. The elimination of heparin itself was slightly retarded by thyroidectomy.     

Lipoprotein lipase activity of rat cardiac muscle. Changes in the enzyme activity during incubations of isolated cardiac-muscle cells in vitro.

1. Isolated cardiac-muscle cells from the hearts of adult rats were shown to retain a high amount of viability during 4 h of incubation when viability was assessed by Trypan Bue stain exclusion and intracellular enzyme leakage. 2. The cells also retained their ability to take up O2 and utilize added substrates over the period of incubation at both 25 and 30 degrees C. 3. When cells from the hearts of fed rats were incubated in a buffered-salts solution at pH 7.4 in the presence of amino acids and heparin, lipoprotein lipase activity in the medium increased progressively. 4. During these incubations the intracellular activity of the enzyme remained constant and the total activity of lipoprotein lipase in the system (cells plus medium) increased by 80% over the 4 h of incubation at 25 degrees C. 5. In the absence of heparin only low amounts of enzyme activity were detectable in the medium and the total lipoprotein lipase activity in the system remained constant. 6. The measurement of lipoprotein lipase activity in either fresh homogenates of the cells or in homogenates of acetone/diethyl ether-dried powders of the cells had no effect on the overall pattern of activity change during the incubations, although as reported previously the total activity detected with acetone/diethyl either-dried preparations was approx. 3-fold higher than with fresh cell homogenates. 7. The observations were compared with published data on lipoprotein lipase activity changes in neonatal heart cell cultures maintained in vitro.     

Lipoprotein lipase activity in neonatal-rat liver cell types.

The lipoprotein lipase activity in the liver of neonatal (1 day old) rats was about 3 times that in the liver of adult rats. Perfusion of the neonatal liver with collagenase decreased the tissue-associated activity by 77%. When neonatal-rat liver cells were dispersed, hepatocyte-enriched (fraction I) and haemopoietic-cell-enriched (fraction II) populations were obtained. The lipoprotein lipase activity in fraction I was 7 times that in fraction II. On the basis of those activities and the proportion of both cell types in either fraction, it was estimated that hepatocytes contained most, if not all, the lipoprotein lipase activity detected in collagenase-perfused neonatal-rat livers. From those calculations it was also concluded that haemopoietic cells did not contain lipoprotein lipase activity. When the hepatocyte-enriched cell population was incubated at 25 degrees C for up to 3 h, a slow but progressive release of enzyme activity to the incubation medium was found. However, the total activity (cells + medium) did not significantly change through the incubation period. Cycloheximide produced a time-dependent decrease in the cell-associated activity. Heparin increased the amount of lipoprotein lipase activity released to the medium. Because the cell-associated activity was unchanged, heparin also produced a time-dependent increase in the total activity. In those cells incubated with heparin, cycloheximide did not affect the initial release of lipoprotein lipase activity to the medium, but blocked further release. The cell-associated activity was also decreased by the presence of cycloheximide in those cells. It is concluded that neonatal-rat hepatocytes synthesize active lipoprotein lipase.     

Effect of ingestion of unsaturated fat on lipolytic activity of rat tissues

Homogenates of some rat tissues, incubated in Tris-maleate buffer containing bovine serum albumin, olive oil emulsion, heparin, and serum, liberated free fatty acids. The total lipolytic activity in tissues of rats fed a low fat, 20% lard, or 20% corn oil diet for 6 wk was measured. Similar activities were found in all the livers, but there was a significant increase in the total lipolytic activity of the mucosa, epididymal fat, and mesenteric tissues after ingestion of an unsaturated fat diet as compared with that containing a more saturated fat. From measurements of the lipolytic activity in the presence of 1 M NaCl or 0.2 M NaF and in the absence and presence of heparin and serum, the conclusion is drawn that more lipoprotein lipase was present in adipose tissue of rats on unsaturated fat diets. An increase in available lipoprotein lipase after unsaturated fat diets may aid in clearing lipids from the blood of these rats and thus in producing the lower blood lipid levels obtained.     

Regulation of lipoprotein lipase. Induction by insulin.

Lipoprotein lipase activity in intact epididymal adipose tissue of fasted rats increased rapidly after treatment with insulin in vivo. In contrast, lipoprotein lipase activity in adipocytes isolated from the contralateral fat pads remained essentially unchanged. When adipocytes were incubated for 30 min at ambient temperature in vitro, about 2 times more lipoprotein lipase activity was found in the medium of cells from insulin-treated rats than in medium from cells of control animals. Following insulin treatment, extracts of tissue acetone powders separated by gel chromatography showed increases in both enzyme activity fractions obtained (designated lipoprotein lipase a and b). However, no consistent differences were observed between fractions derived from adipocyte acetone powders of insulin-treated and control animals. All the observed effects of insulin on lipoprotein lipase activity were abolished by cycloheximide treatment in vivo. These data indicate that following insulin treatment, increased lipoprotein lipase activity in adipose tissue results from enhanced enzyme secretion by the fat cell and subsequent accumulation in the tissue, thus implicating the adipocyte secretory mechanism as a major site of regulation of lipoprotein lipase activity in adipose tissue.     

Lipoprotein lipase in liver. Release by heparin and immunocytochemical localization

We have previously demonstrated that infusion of Intralipid to rats causes a pronounced increase of the lipoprotein lipase activity in the liver. In this paper we study where in the liver this lipoprotein lipase is located. When isolated livers from Intralipid-treated rats were perfused with heparin, substantial amounts of lipoprotein lipase were released into the perfusate. The identity of the lipase activity was demonstrated by specific inhibition with antisera to lipoprotein lipase, and to hepatic lipase, respectively, and by separation of the two lipase activities by chromatography on heparin-Sepharose. We have also studied the localization of both enzymes by an immunostaining procedure based on post-embedding incubation of ultrathin tissue sections with specific antibodies which were then visualized using protein A-colloidal gold complexes. There was no marked difference in localization for the two enzymes which were both seen at the luminal side of endothelial cells, at the interdigitations of the space of Disse and inside both hepatocytes and endothelial cells. Thus, lipoprotein lipase is present in the liver in positions similar to where the functional pool of hepatic lipase is located and analogous to where lipoprotein lipase is found in extrahepatic tissues. These results raise the possibility that the enzyme has a functional role in the liver.     

Selective release of extrahepatic lipoprotein lipase by polymetaphosphate

We have studied the lipase released into the circulation by polymetaphosphate injection into rats. Lipase release was in proportion to the dose injected. The post-polymetaphosphate lipase was almost completely inhibited by high salt concentrations or by addition of protamine sulfate to the assay system suggesting that this compound released lipoprotein lipase and not hepatic triglyceride lipase. The lipases released by polymetaphosphate and by heparin were compared using a heparin-sepharose affinity column technique which separates lipoprotein lipase from hepatic triglyceride lipase. While heparin released both lipoprotein lipase and hepatic triglyceride lipase, polymetaphosphate released almost exclusively lipoprotein lipase. Other experiments showed that neither polymetaphosphate nor heparin inhibited the hepatic lipase when added to the assay. These results suggest that lipoprotein lipase may be released by the negative charge on these high-charge polymers while hepatic triglyceride lipase release may require the specific sugar configuration of heparin.     

Lipoprotein lipase activity of rat cardiac muscle. The intracellular distribution of the enzyme between fractions prepared from cardiac muscle and cells isolated from the hearts of fed and starved animals.

1. Subcellular fractions, characterized by using morphological, compositional and enzymic markers, were prepared from rat heart tissue and cells isolated from the hearts of fed and 24 h-starved rats. 2. The lipoprotein lipase activity of fractions from whole tissue and isolated cells was determined in either fresh fractions or in acetone/diethyl ether powders of the fractions. 3. Lipoprotein lipase activity was present in all the fractions from tissue and cells, but was found to be of highest relative specific activity in the microsomal () fractions. 4. In fractions prepared from the isolated cells of hearts from starved rats the proportion of the total lipoprotein lipase present and its relative specific activity in the microsomal fraction were greater than in the equivalent fractions from fed animals. 5. The enhancement of lipoprotein lipase activity as a result of the acetone/diethyl ether powder preparation of fractions was most extensive in the microsomal fractions. 6. Investigation of the microsomal fraction showed that the lipoprotein lipase activity present was in two pools, one of which was within endoplasmic-reticulum vesicles. 7. The observations were consistent with the possibility that the cardiac-muscle cell could be the origin of the lipoprotein lipase activity functional in triacylglycerol uptake by the heart.     

Effect of dietary palm oil on lipoprotein lipases: lipoprotein levels and tissue lipids in rat

The aims of our study were to investigate the effect of dietary palm oil on the levels of lipoprotein lipase, hepatic lipase, fat distribution (in the aorta and liver), and total cholesterol, HDL, LDL, and triacylglycerol levels in young rats (70 g body wt) over a period of 10 weeks. Palm oil-fed rats showed higher growth rate and lower triacylglycerol levels than the control group. Hepatic lipase activity was correlated to the liver fat distribution (correlation coefficient, r = +0.682) as seen by histopathological sections and was similar for both the palm oil and the control diets. Palm oil-fed rats exhibited a significantly higher HDL cholesterol to total plasma cholesterol ratio when compared to animals fed the control diet. The triacylglycerol levels correlated inversely to the HDL cholesterol levels (r = -0.536) while the lipoprotein lipase (LPL) activity correlated directly to the LDL level (r = +0.617) for both groups of animals. The fatty acid profiles of adipose and liver tissues and plasma revealed that saturated fatty acids--palmitic and stearic--were preferentially incorporated in liver and adipose tissues and less in the plasma. This accounts for lack of deposition in the arterial wall and for the antithrombotic tendency of palm oil. Thus, our present findings suggest that dietary palm oil may not contribute to the risk for coronary heart disease.     

Release of lipoprotein lipase to plasma by triacylglycerol emulsions. Comparison to the effect of heparin.

It was previously known that lipoprotein lipase (LPL) activity in plasma rises after infusion of a fat emulsion. To explore the mechanism we have compared the release of LPL by emulsion to that by heparin. After bolus injections of a fat emulsion (Intralipid) to rats, plasma LPL activity gradually rose 5-fold to a maximum at 6-8 min. During the same time the concentration of injected triacylglycerols (TG) decreased by about half. Hence, the time-course for plasma LPL activity was quite different from that for plasma TG. The disappearance of injected 125I-labelled bovine LPL from circulation was retarded by emulsion. This effect was more marked 30 min than 3 min after injection of the emulsion. The data indicate that the release of LPL into plasma is not solely due to binding of the lipase to the emulsion particles as such, but involves metabolism of the particles. Emulsion increased the fraction of labelled LPL found in adipose tissue, heart and the red muscle studied, but had no significant effect on the fraction found in liver. The effects of emulsion were quite different from those of heparin, which caused an immediate release of the lipase to plasma, decreased uptake of LPL in most extrahepatic tissues by 60-95%, and increased the fraction taken up in the liver.     

Endogenous plasma lipoprotein lipase activity in fed and fasting rats may reflect the functional pool of endothelial lipoprotein lipase

In this study, a correlation was sought between the circulating lipoprotein lipase activity and nutritional state in the rat. In fed rats, the plasma lipoprotein lipase activity was between 30 and 120 munits/ml, whereas after an overnight fast in restraining cages, the lipoprotein lipase plasma levels were between 280 and 500 munits/ml. The plasma lipoprotein lipase activity was inhibited by a specific high titre goat antiserum to rat lipoprotein lipase. No effect of fasting was seen on the plasma hepatic triacylglycerol lipase. 6 h after fasting, adipose tissue lipoprotein lipase decreased maximally, but plasma lipoprotein lipase was not changed and rose only after 16 h. Thus, it seems that most of the lipoprotein lipase activity in the fasting plasma was related to the 3-fold rise in lipoprotein lipase activity in the heart, which may represent total muscle lipoprotein lipase. The increase in heart lipoprotein lipase was due in part to an increase in the t1/2 of the enzyme from 1.2 to 2.9 h. To determine whether the high plasma levels in the fasting rats might result from impaired clearance of the enzyme by the liver, functional hepatectomy was carried out. 15 min after hepatectomy, plasma lipoprotein lipase rose up to 20-fold in fed and about 6-fold in fasting rats. Lipoprotein lipase activity extracted by the liver was calculated to be 30-60 munits/ml in the fed and 171-247 munits/ml plasma per min in fasting rats. An increase in lipoprotein lipase activity in extrahepatic tissues (heart, lung, kidney, diaphragm and adrenal) occurred 30 min after hepatectomy in fed rats. The increase in heart lipoprotein lipase was due to an increase in heparin-releasable fraction. Since no impairment of hepatic clearance of circulating plasma lipoprotein lipase was found, the high fasting plasma lipoprotein lipase activity may be related to an increase in enzyme synthesis, decreased enzyme turnover and an expansion of the functional pool in tissues such as the heart and probably muscle. The present findings indicate that measurement of endogenous plasma lipoprotein lipase can provide information with respect to the size of the functional pool under normal and pathological conditions.     

Mechanism of alteration of the functional fraction of lipoprotein lipase in rat heart

Feeding glucose to fasted rats resulted in a decrease in the activity of heparin-releasable lipoprotein lipase in heart perfusates. Upon feeding fat to glucose-fed animals the level of heparin-releasable lipoprotein lipase increased 10–14 fold. An immunological titration was used to determine whether the changes in lipase activity following the various nutritional treatments were due to changes in the amount of enzyme present or to activation/inactivation processes. These data suggest that changes in the enzyme activity are due to alteration in the quantity of lipoprotein lipase protein.     

Post-heparin triacylglycerol lipases in ovine plasma

Lipoprotein lipase and hepatic lipase have been shown to be present in the post-heparin plasma of sheep. Intravenous injection of heparin into sheep produced a rapid increase in the free fatty acid concentration and lipolytic enzyme activity of the plasma, both peaking within 5-15 min and then falling to pre-heparin levels within 30-60 min. Lipolytic activity was not detected in plasma before heparin treatment. Two distinct lipolytic activities were separated from the plasma by chromatography on heparin-Sepharose 6B. Lipoprotein lipase was identified on the basis that the lipolytic activity was dependent upon the addition of plasma, inhibited by 1M NaCl, and inhibited by a specific antiserum against lipoprotein lipase. The second lipolytic activity of plasma was identified as hepatic lipase, as it was not dependent upon plasma for activity, nor was it inhibited by 1M NaCl or antiserum against lipoprotein lipase. Its properties were identical to the lipase extracted from the liver of sheep. Lipoprotein-lipase activity, but not hepatic-lipase activity, was dependent upon the nutritional state of the sheep at the time of heparin injection. However, hepatic lipase comprised a significant proportion of the total lipolytic activity.     

The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339.

1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.     

Influence of heparin on the removal of serum lipoprotein lipase by the perfused liver of the rat

Isolated rat livers were perfused with whole rat blood containing postheparin lipoprotein lipase (LPL) activity. LPL activity disappeared rapidly from the perfusate; the extraction ratio (portal vein-hepatic vein difference) was 0.70 for all time periods studied. Control experiments established that the disappearance of LPL was not due to non-specific inactivation in the apparatus or to the release of an inhibitory by the liver. The addition of heparin to the perfusate in suitable concentration (4 units/ml) almost completely blocked the disappearance of LPL activity from the perfusate. In addition to the perfusion experiments, we studied the effect of heparin on LPL activity when added to the LPL assay system. When heparin was added to the assay system containing fresh postheparin serum from rats, it stimulated LPL activity by about 70%. When heparin was added to postheparin serum which had been perfused through the liver, it stimulated LPL activity over 200%, but it did not restore LPL to its preperfusion value. These observations are compatible with a two-step inactivation system for LPL by the liver. The first step may involve a dissociation of a heparin-apoenzyme complex followed by destruction of the heparin. The second step may involve the removal of the apoenzyme of LPL.     

Lipoprotein lipase in rat lung. Effect of dexamethasone.

The effect of hormone administration on the activity of lipoprotein lipase in the lung was studied in the rat. The following hormones were administered: dexamethasone, L-thyroxine, estradiol-17beta and progesterone. In addition, lung lipoprotein lipase activity was studied in diabetic and lactating rats. Lipoprotein lipase activity was measured in dried, defatted preparations of rat lung using double labeled ([14C]palmitate, [3H]glycerol) chylomicron triacylglycerol as substrate. Dexamethasone administration caused a rise of 70% in the level of activity of lipoprotein lipase in acetone powders of lung and a 100% increase in the amount of enzyme released during heparin infusion into isolated, perfused lungs. Enzyme activity was higher in lungs of females than of male rats; however; the level of activity was unaffected by estrogen or progesterone administration to either male or ovariectomized rats. Diabetes, hyperthyroidism or lactation did not change lipoprotein lipase activity in the lung. The constant presence of lipoprotein lipase activity in the lung suggests that this organ is able to maintain a steady supply of triacylglycerol-fatty acids under a variety of physiological and pathological conditions. Stimulation of enzyme activity by dexamethasone could lead to increased uptake of triacylglycerol-fatty acids by the lung and may thus be a contributing factor to corticosteroid-induced enhanced surfactant synthesis.